The objective of this proposal is to obtain support for the continuation of our studies on the assembly of glycosylphosphatidylinositols (GPIs) and GPI-anchored proteins. The latter include functionally important proteins such as cell surface receptors, cell adhesion molecules and protozoal surface antigens. A deficiency in GPI biosynthesis in hematopoietic cells causes paroxysmal nocturnal hemoglobinuria (PNH), an acquired hemolytic disease in humans characterized by abnormal activation of complement on erythrocytes due to a deficiency of GPI-anchored complement regulatory proteins. Genetic abrogation of GPI biosynthesis results in embryonic lethality in mammals. The aims of this proposal are to understand aspects of GPI anchor biosynthesis with the overall objective of contributing to efforts to manipulate and control the GPI pathway. Such efforts are central to the development of anti-protozoal and anti-fungal drugs, as well to the possible treatment of human diseases in which GPI-anchored proteins play a key part. Our specific aims are to analyze GPI N-acetyl-glucosaminyl-transferase (GPI-GnT) and GPI transamidase (GPT), the first and last enzymes of the assembly pathway, and to pursue the novel finding that early enzymes of the GPI assembly pathway are localized to a mitochondria-associated ER membrane domain (MAM). Both GPI-GnT and GPT are novel, multi-subunit complexes: GPI-GnT is responsible for the synthesis of the first GPI biosynthetic intermediate, N-acetylglucosaminylphosphatidylinositol, and GPT is the enzyme that attaches GPI anchors to proteins. We will define the subunit composition of the yeast GPI-GnT complex and characterize its sugar nucleotide and phospholipid binding components using photoaffinity labeling, site-directed mutagenesis, and fluorescence resonance energy transfer. We will similarly define human GPT, focusing on the composition of the complex, subunit interactions required for complex assembly and the targeting motifs needed for endoplasmic reticulum localization. The MAM domain represents an unusual heterogeneity within the ER that, through sequestration of certain of the GPI biosynthetic enzymes, may contribute to the regulation of GPI biosynthesis. We will focus on PIG-L, the second enzyme of the pathway, whose activity is known to be localized to the MAM. We will use truncated and chimeric PIG-L constructs, subcellular fractionation, fluorescence and electron microscopy, and single cell fluorescence photobleaching techniques to study the targeting of PIG-L to the MAM and characterize its dynamics within the MAM.